Anti-ophidian activity of Bredemeyera floribunda Willd. (Polygalaceae) root extract on the local effects induced by Bothrops jararacussu venom

Bredemeyera floribunda roots are popularly used to treat snakebites in the semiarid region of Northeast Brazil, and previous studies indicate the anti-ophidian actions of triterpenoid saponins found in its roots. To assess B. floribunda root extract (BFRE) activity against the effects of Bothrops jararacussu venom (BjuV), antiphospholipasic, antiproteolytic, antihemorrhagic, antinecrotic, and anti-edematogenic activities were investigated in mice. Phytochemical analysis revealed the presence of saponins, flavonoids, and sugars, with rutin and saccharose being the major constituents of BFRE. Acute toxicity was determined and BFRE was nontoxic to mice. Phospholipase A2 and proteolytic activities induced by BjuV were inhibited in vitro by BFRE at all concentrations tested herein. BFRE (150 mg/kg) inhibited paw edema induced by BjuV (50 µg/animal), reducing total edema calculated by area under the curve, but carrageenan-induced paw edema was unchanged. Hemorrhagic and necrotizing actions of BjuV (50 µg/animal) were considerably decreased by BFRE treatment. Thus, BFRE blocked the toxic actions of B. jararacussu venom despite having no anti-inflammatory activity, which points to a direct inhibition of venom's toxins, as demonstrated in the in vitro assays. The larger amounts of rutin found in BFRE may play a role in this inhibition, since 3′,4′-OH flavonoids are known inhibitors of phospholipases A2.

Renal- and calcium-dependent vascular effects of Polybia paulista wasp venom

In the present study, the effects of Polybia paulista venom (PPV) on renal and vascular tissues were investigated. Isolated kidneys perfused with PPV (1 and 3 ìg/mL) had increased perfusion pressure, renal vascular resistance, urinary flow, and glomerular filtration rate; and reduced sodium tubular transport. Histological evaluation demonstrated deposits of proteins in Bowman's space and tubular lumen, and focal areas of necrosis. The venom promoted a cytotoxic effect on Madin-Darby canine kidney (MDCK) cells. A significant increase in lactic dehydrogenase levels was observed in response to venom exposure. In isolated mesenteric vascular beds, pressure and vascular resistance augmented in a dose-dependent manner. PPV increased the contractility of aortic rings maintained under basal tension. This contractile response was inhibited when preparations were maintained in Ca2+-free medium. Likewise, verapamil, a voltage-gated calcium channel blocker, also inhibited the contractile response. In this study, phentolamine, a blocker of á-adrenergic receptor blocker, significantly reduced the contractile effect of PPV in the aortic ring. In conclusion, PPV produced nephrotoxicity, which suggests a direct effect on necrotic cellular death in renal tubule cells. The vascular contractile effect of PPV appears to involve calcium influx through voltage-gated calcium channels via adrenergic regulation.

Renal and vascular effects of Crotalus durissus cumanensis venom and its crotoxin fraction

In this study, we evaluated the actions of Crotalus durissus cumanensis venom (CDCmV), and its crotoxin (Crtx) fraction, on renal and vascular functions in Wistar rats. In isolated perfused kidneys, CDCmV (10 µg/mL) significantly increased the perfusion pressure (PP) from 110.7 ± 2.4 to 125.3 ± 2.8 mmHg after 30 minutes. This effect was accompanied by an increased renal vascular resistance (RVR) from 5.4 ± 0.1 to 6.2 ± 0.2 mmHg/mL.g-1.min-1. We observed decreases in urinary flow (UF) from 0.13 ± 0.01 to 0.05 ± 001 mL.g-1.min-1 and glomerular filtration rate (GFR) from 0.66 ± 0.06 to 0.18 ± 0.02 mL.g-1.min-1. Crtx did not change PP or RVR, but diminished GFR (from 0.65 ± 0.05 to 0.26 ± 003 mL.g-1.min-1) and UF (from 0.11 ± 0.008 to 0.09 ± 0.008 mL.g-1.min-1). Both CDCmV and Crtx reduced the percentage of tubular transport of sodium, chloride and potassium. The cytotoxicity of these substances against MDCK cells was tested by the MTT method: only CDCmV caused a decrease in the cell viability with an IC50 of 5.4 µg/mL. In endothelium-intact isolated aortic rings, CDCmV (0.1 to 30 µg/mL) increased the sustained phenylephrine-induced contraction to a value of 130.0 ± 6.6 percent of its corresponding control, but showed a relaxant effect in endothelium-denuded preparations. Similar results were observed in aortic rings contracted with potassium (40 mM). Crtx was ineffective in aortic ring assays. Thus, it is reasonable to suggest that the renal effects induced by the CDCmV may be due to its influence on the endothelium's ability to release factors that can alter the contractile behavior of vascular smooth muscle. In conclusion, CDCmV is toxic to kidney cells. It changes parameters of the renal function including the glomerular filtration rate, renal vascular resistance and tubular transport. The actions induced by CDCmV also involve endothelium-dependent vasoactive properties. Their effects may be only partially attributed to Crtx.

Purification and biological effects of a C-type lectin isolated from bothrops moojeni

Snake venom proteins from the C-type lectin family have very distinct biological activities despite their highly conserved primary structure, which is homologous to the carbohydrate recognition region of true C-type lectins. We purified a lectin-like protein (BmLec) from Bothrops moojeni venom and investigated its effect on platelet aggregation, insulin secretion, antibacterial activity, and isolated kidney cells. The BmLec was purified using two chromatographic steps: affinity chromatography and reverse phase high performance liquid chromatography (HPLC). BmLec showed a dose-dependent platelet aggregation and significantly decreased the bacterial growth rate in approximately 15 percent. During scanning electron microscopy, the profile of Xanthomonas axonopodis pv. passiflorae treated with lectin disclosed a high vesiculation and membrane rupture. BmLec induced a strong and significant increase in insulin secretion at 2.8 and 16.7 mM glucose concentrations, and this effect was seen in the presence of EGTA in both experiments. BmLec (10 µg/mL) increased the perfusion pressure, renal vascular resistance and urinary flow. The glomerular filtration rate and percentages of sodium, potassium and chloride tubular transport were reduced at 60 minutes of perfusion. Renal alterations caused by BmLec were completely inhibited by indomethacin in all evaluated parameters. In conclusion, the C-type lectin isolated from Bothrops moojeni affected platelet aggregation, insulin secretion, antibacterial activity and isolated kidney function.

Antimicrobial activity of an L-amino acid oxidase isolated from Bothrops leucurus snake venom

Some snake venom proteins present enzymatic activities, such as L-amino acid oxidase (LAAO). The aim of this paper was to investigate the effect of Bothrops leucurus total venom (BleuTV) and its fraction LAAO (BleuLAAO) on bacteria, yeast, and promastigote forms of Leishmania amazonensis and Leishmania chagasi, and epimastigote forms of Trypanosoma cruzi. BleuTV was isolated with a Protein Pack 5PW® (Waters Corporation, USA), and several fractions were obtained. BleuLAAO was purified to high molecular homogeneity, and its N-terminal amino acid sequence shared a high degree of amino acid conservation with other LAAOs. BleuTV inhibited Staphylococcus aureus growth in a dose-dependent manner, with a minimum inhibitory concentration (MIC) of 25 ìg/mL, which corresponded to its minimum lethal concentration (MLC). BleuTV also inhibited the growth of promastigote forms of L. chagasi and L. amazonensis, with respective IC50 values of 1.94 ìg/mL and 5.49 ìg/mL. Furthermore, it repressed T. cruzi growth with an IC50 of 1.14 ìg/mL. However, BleuLAAO did not inhibit the growth of the microorganisms studied and was not toxic to macrophages. BleuTV had low toxicity against macrophages at the concentrations studied. In conclusion, whole venom from Bothrops leucurus inhibited the growth of some microorganisms, including S. aureus, Leishmania sp., and T. cruzi.

Purification and n-terminal sequencing of two presynaptic neurotoxic PLA2, neuwieditoxin-I and neuwieditoxin-II, from Bothrops neuwiedi pauloensis (jararaca pintada) venom

Two presynaptic phospholipases A2 (PLA2), neuwieditoxin-I (NeuTX-I) and neuwieditoxin-II (NeuTX-II), were isolated from the venom of Bothrops neuwiedi pauloensis (BNP). The venom was fractionated using molecular exclusion HPLC (Protein-Pak 300SW column), followed by reverse phase HPLC (æBondapak C18 column). Tricine-SDS-PAGE in the presence or absence of dithiothreitol showed that NeuTX-I and NeuTX-II had a molecular mass of approximately 14 kDa and 28kDa, respectively. At 10æg/ml, both toxins produced complete neuromuscular blockade in indirectly stimulated chick biventer cervicis isolated preparation without inhibiting the response to acetylcholine, but NeuTX-II reduced the response to KCl by 67.0±8.0 percent (n=3; p<0.05). NeuTX-I and NeuTX-II are probably responsible for the presynaptic neurotoxicity of BNP venom in vitro. In fact, using loose patch clamp technique for mouse phrenic nerve-diaphragm preparation, NeuTX-I produced a calcium-dependent blockade of acetylcholine release and caused appearance of giant miniature end-plate potentials (mepps), indicating a pure presynaptic action. The N-terminal sequence of NeuTX-I was DLVQFGQMILKVAGRSLPKSYGAYGCYCGWGGRGK (71 percent homology with bothropstoxin-II and 54 percent homology with caudoxin) and that of NeuTX-II was SLFEFAKMILEETKRLPFPYYGAYGCYCGWGGQGQPKDAT (92 percent homology with Basp-III and 62 percent homology with crotoxin PLA2). The fact that NeuTX-I has Q-4 (Gln-4) and both toxins have F-5 (Phe-5) and Y-28 (Tyr-28) strongly suggests that NeuTX-I and NeuTX-II are Asp49 PLA2.

Crotacetin, a novel snake venom C-type lectin, is homolog of convulxin

Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins, which are able to interfere with hemostasis. They share significant similarity in their primary structures with C-type lectins of other animals, and also present a conserved carbohydrate recognition domain (CRD). A very well studied sv C-type lectin is the heterodimeric toxin, convulxin (CVX), from the venoms of South American rattlesnakes, Crotalus durissus terrificus and C. d. cascavella. It consists of two subunits, alfa (CVXa, 13.9 kDa) and beta (CVXb, 12.6 kDa), joined by inter and intra-chain disulfide bounds, and is arranged in a tetrameric a4b4 conformation. Convulxin is able to activate platelet and induce their aggregation by acting via p62/GPVI collagen receptor. Several cDNA precursors, homolog of CVX subunits, were cloned by PCR homology screening. As determined by computational analysis, one of them, named crotacetin b subunit, was predicted as a polypeptide with a tridimensional conformation very similar to other subunits of convulxin-like snake toxins. Crotacetin was purified from C. durissus venoms by gel permeation and reverse phase high performance liquid chromatography. The heterodimeric crotacetin is expressed in the venoms of several C. durissus subspecies, but it is prevalent in the venom of C. durissus cascavella. As inferred from homology modeling, crotacetin induces platelet aggregation but noticeably exhibits antimicrobial activity against Gram-positive and Gram-negative bacteria

The pharmacological effect of Bothrops neuwiedii pauloensis (jararaca-pintada) snake venom on avian neuromuscular transmission

The neuromuscular effects of Bothrops neuwiedii pauloensis (jararaca-pintada) venom were studied on isolated chick biventer cervicis nerve-muscle preparations. Venom concentrations of 5-50 æg/ml produced an initial inhibition and a secondary increase of indirectly evoked twitches followed by a progressive concentration-dependent and irreversible neuromuscular blockade. At venom concentrations of 1-20 æg/ml, the responses to 13.4 mM KCl were inhibited whereas those to 110 æM acetylcholine alone and cumulative concentrations of 1 æM to 10 mM were unaffected. At venom concentrations higher than 50 æg/ml, there was pronounced muscle contracture with inhibition of the responses to acetylcholine, KCl and direct stimulation. At 20-24ºC, the venom (50 æg/ml) produced only partial neuromuscular blockade (30.7 ± 8.0 percent, N = 3) after 120 min and the initial inhibition and the secondary increase of the twitch responses caused by the venom were prolonged and pronounced and the response to KCl was unchanged. These results indicate that B.n. pauloensis venom is neurotoxic, acting primarily at presynaptic sites, and that enzyme activity may be involved in this pharmacological action